The five most intense peptide ions were fragmented by collision-induced dissociation with multistage activation of the neutral loss of phosphoric acid from the parent ion (neutral loss masses = 49, 32.33 and 24.5 for z = 2,3 and 4).
Samples were analysed on an LTQ-Orbitrap XL mass spectrometer as described above, except that the top five ions were fragmented in the linear ion-trap using multistage activation of the neutral loss of phosphoric acid from the parent ion (neutral loss masses=49, 32.33 and 24.5 for z=2, 3 and 4 respectively).
In the LTQ-Orbitrap mass spectrometer, a survey scan was performed over a split mass range (m/ z 300 800 and 800 2000) in the Orbitrap analyser each of them triggering five MS LTQ acquisitions of the five most intense ions using multistage activation on the neutral loss of 98, 49 and 32.33 Da.
Precursors were analysed in the orbitrap set to a resolution of 60 000 and tandem MS/MS was performed in the LTQ with multistage activation (MSA) of the precursors and precursor −98/z, as described previously (Schroeder et al, 2004).
To ensure the best fragmentation spectrum for any phosphopeptide was obtained, the precursors were fragmented using multistage activation (MSA) of the precursor mass (m/z) minus 49, 32.33 and 24.25 Da (this represents the loss of H3PO4 from a 2+, 3+ or 4+ precursor ion).
These peptides were analyzed similarly except the usage of multistage activation in the fragmentation method.
The gradient was 150 min from 2 to 40%% of mobile phase B and multistage activation was enabled with neural losses of 24.49, 32.66, 48.999, 97.97, 195.94, and 293.91 Da for the 10 most intense precursor ions.
Multistage activation (MSA) for neutral losses of 98.0, 49.0, and 32.33 Da was enabled to enhance fragmentation of phosphorylated peptides.
Multistage activation (MSA) was used for enhanced fragmentation of putative phosphate group containing ions as described (Basell et al. 2014).
Multistage activation was used to provide an MS scan of parent ions showing a neutral loss of 48.9885, 32.6570 and 24.4942, allowing for 2+, 3+ and 4+ ions respectively.
CID was triggered first in normal mode and second with the multistage activation option on when the prominent neutral loss fragment of −172 Da was observed as most abundant ion.
